Stability and Strand Asymmetry in the Non-B DNA Structure at the bcl-2 Major Breakpoint Region

Abstract

The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.